In multicellular organisms, such as mammals, cells communicate with each other by signal transduction pathways, in which a secreted ligand (e.g. cytokines, growth factors, or hormones) binds to its cell surface receptor(s), leading to receptor activation. The receptors are membrane proteins, which consist of an extracellular domain responsible for ligand binding, a central transmembrane region followed by a cytoplasmic domain responsible for sending the signal downstream. Signal transduction can take place in the following three ways: paracrine (communication between neighboring cells), autocrine (cell communication to itself) and endocrine (communication between distant cells through circulation), depending on the source of a secreted signal and the location of target cell expressing a receptor(s). One of the general mechanisms underlying receptor activation, which sets off a cascade of events beneath the cell membrane including the activation of gene expression, is that a polypeptide ligand such as a cytokine, is present in an oligomeric form, such as a homo-dimer or trimer, which when bound to its monomeric receptor at the cell outer surface, leads to the oligomerization of the receptor. Signal transduction pathways play a key role in normal cell development, differentiation and immune surveillance against cancer, as well as in response to external insults such as bacterial and viral infections. Abnormalities in such signal transduction pathways, in the form of either underactivation (e.g. lack of ligand) or overactivation (e.g. too much ligand), are the underlying causes for pathological conditions and diseases such as arthritis, cancer, AIDS, and diabetes.
One of the current strategies for treating these debilitating diseases involves the use of receptor decoys, such as soluble receptors consisting of only the extracellular ligand-binding domain, to intercept a ligand and thus overcome the overactivation of a receptor. A good example of this strategy is the creation of Enbrel®, a dimeric soluble TNF-α receptor-immunoglobulin (IgG) fusion protein by Immunex, which is now part of Amgen. Anti-TNF-α biologics have now become the standard of care for a host of autoimmune diseases. The TNF family of cytokines is one of the major pro-inflammatory signals produced by the body in response to infection and tissue injury. However, abnormal production of these cytokines, for example, in the absence of infection or tissue injury, has been shown to be one of the underlying causes for diseases such as arthritis and psoriasis. Naturally, a TNF-α receptor is present in monomeric form on the cell surface before binding to its ligand, TNF-α, which exists, in contrast, as a homotrimer. Accordingly, fusing a soluble TNF-α receptor with the Fc region of immunoglobulin G1, which is capable of spontaneous dimerization via disulfide bonds, allowed the secretion of a dimeric soluble TNF-α receptor. In comparison with the monomeric soluble receptor, the dimeric TNF-α receptor II-Fc fusion has a greatly increased affinity to the homo-trimeric ligand. This provides a molecular basis for its clinical use in treating rheumatoid arthritis (RA), an autoimmune disease in which constitutively elevated TNF-α, a major pro-inflammatory cytokine, plays an important causal role.
In contrast, abnormalities in the production of certain TNF family of cytokines seems to be linked to failure in immune surveillance against cancer. In fact the founding member, TNF-α was initially identified as a cancer cell killer and so named as TNF (tumor necrosis factor). Now, it is known that several members of TNF family of cytokines, including TNF-α, FasL and TRAIL/Apo2L, have the ability to potently elicit cancer cell killing via either apoptosis or necrosis. However, it turns out that overexpression of TNF-α and FasL are toxic to mammals as they elicit potent inflammatory responses. So far only TRAIL/Apo2L retains the characteristics for being cancer-specific killer via apoptosis and has been extensively studied as an anti-cancer biologics (Wang and El-Deiry, 2003). Recombinant TRAIL/Apo2L (Dulanermin/AMG 951) produced from E. coli by Amgen and Genentech showed promising results in preclinical xenograft animal models (Kelley et al., 2001) as well as in Phase I clinical trials against multiple cancers (Soria et al., 2010). However, it failed in several Phase II trials due to lack of efficacy albeit good safety profile (Soria et al., 2011). The lack of efficacy has been linked to Dulanermin's poor pharmacokinetic profile, with very short systemic half-life in mammals; this is likely due to its small molecular weight (˜60 kDa) and the instability of its non-covalently linked trimeric structure, both ultimately leading to its rapid elimination via renal filtration (Kelley et al., 2001). Thus, trimerization via covalent bond linkage may stabilize TRAIL/Apo2L trimeric structure essential for its biological activity as well as to increase the molecular weight to extend half-life for improved efficacy.
Although there have been various approaches to overcoming such challenges faced by native TRAIL (de Miguel et al. 2016), such attempts have ultimately fallen short as feasible human therapies. A leucine zipper fused TRAIL and a cross-linked TRAIL stabilized by inserted mutations creating an additional artificial disulfide bond are both potentially immunogenic in humans due to the non-human nature of their added oligomerization domains; moreover, both have demonstrated hepatotoxicity not observed with native TRAIL. Other methods for extending half-life of TRAIL have also encountered obstacles; albumin conjugated TRAIL nanoparticles must be prepared in organic solvents and production was extremely limited. Similar production issues are faced by liposome conjugated TRAIL. Recently, immuno-oncology as a paradigm for cancer therapy has made great progress with the approvals of therapeutic antibodies against PD-1, PD-L1 and CTLA-4, with exciting sustained remissions for certain cancer patients, including melanoma. Programmed cell death protein 1, also known as PD-1, is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2. PD-1 is a member of the extended CD28/CTLA-4 family of T cell regulators. PD-1 and its ligands play an important role in downregulating the immune system by preventing the activation of T-cells, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is accomplished through a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells). Therapeutic antibodies that block PD-1 or its ligand (PD-L1) as well as CTLA-4, activate the immune system to attack tumors and are therefore used to treat cancer. These breakthroughs demonstrated that cancer is an immune disease with cancer cells evading the body's immune surveillance by inhibiting several key immune check-points such as PD-1 and CTLA-4 signaling pathways. Importantly, there is an increasing body of evidence showing that several members of TNF family of cytokines, such as 4-1BBL (also called CD137L) and OX40L, can potently stimulate proliferation and survival of antigen-specific cytotoxic T cells as well as stimulate the memory T cell response. Thus, combinational use of PD-1 or CTLA-4 antagonists with 4-1BB or OX40 agonists may fully activate the body's immune attack against cancer. Like all members of TNF family of cytokines, 4-1BBL and OX40L are small in molecular weight and homo-trimeric in structure without inter-subunit disulfide bond linkages.
Clearly, there is a great need to be able to create secreted homo-trimeric and disulfide bond-strengthened TNF family of cytokines that retain full biological activities. Compared to their native counterparts produced from bacteria, such rationally designed TNF family of ligand-Trimers produced in mammalian cells such as CHO cells, may significantly increase the efficacy for cancer treatment. To be therapeutically feasible, a desired trimerizing protein moiety for biologic drug designs should satisfy the following criteria. Ideally it should be part of a naturally secreted protein, like immunoglobulin Fc, that is also abundant (non-toxic) in the circulation, human in origin (lack of immunogenicity), relatively stable (long half-life) and capable of efficient self-trimerization which is strengthened by inter-chain covalent disulfide bonds, so the trimerized TNF family of cytokines are structurally stable.
Collagen is a family of fibrous proteins that are the major components of the extracellular matrix. It is the most abundant protein in mammals, constituting nearly 25% of the total protein in the body. Collagen plays a major structural role in the formation of bone, tendon, skin, cornea, cartilage, blood vessels, and teeth. The fibrillar types of collagen I, II, III, IV, V, and XI are all synthesized as larger trimeric precursors, called procollagens, in which the central uninterrupted triple-helical domain consisting of hundreds of “G-X-Y” repeats (or glycine repeats) is flanked by non-collagenous domains (NC), the N-propeptide and the C-propeptide. Both the C- and N-terminal extensions are processed proteolytically upon secretion of the procollagen, an event that triggers the assembly of the mature protein into collagen fibrils which forms an insoluble cell matrix. BMP-1 is a protease that recognizes a specific peptide sequence of procollagen near the junction between the glycine repeats and the C-prodomain of collagens and is responsible for the removal of the propeptide. The shed trimeric C-propeptide of type I collagen is found in human sera of normal adults at a concentration in the range of 50-300 ng/mL, with children having a much higher level which is indicative of active bone formation. In people with familial high serum concentration of C-propeptide of type I collagen, the level could reach as high as 1-6 μg/mL with no apparent abnormality, suggesting the C-propeptide is not toxic. Structural study of the trimeric C-propeptide of collagen suggested that it is a tri-lobed structure with all three subunits coming together in a junction region near their N-termini to connect to the rest of the procollagen molecule. Such geometry in projecting proteins to be fused in one direction is similar to that of Fc dimer.
Type I, IV, V and XI collagens are mainly assembled into heterotrimeric forms consisting of either two α-1 chains and one α-2 chain (for Type I, IV, V), or three different a chains (for Type XI), which are highly homologous in sequence. The type II and III collagens are both homotrimers of α-1 chain. For type I collagen, the most abundant form of collagen, stable α(I) homotrimer is also formed and is present at variable levels in different tissues. Most of these collagen C-propeptide chains can self-assemble into homotrimers, when over-expressed alone in a cell. Although the N-propeptide domains are synthesized first, molecular assembly into trimeric collagen begins with the in-register association of the C-propeptides. It is believed the C-propeptide complex is stabilized by the formation of interchain disulfide bonds, but the necessity of disulfide bond formation for proper chain registration is not clear. The triple helix of the glycine repeats and is then propagated from the associated C-termini to the N-termini in a zipper-like manner. This knowledge has led to the creation of non-natural types of collagen matrix by swapping the C-propeptides of different collagen chains using recombinant DNA technology. Non-collagenous proteins, such as cytokines and growth factors, also have been fused to the N-termini of either pro-collagens or mature collagens to allow new collagen matrix formation, which is intended to allow slow release of the noncollagenous proteins from the cell matrix. However, under both circumstances, the C-propeptides are required to be cleaved before recombinant collagen fibril assembly into an insoluble cell matrix.
Although, other protein trimerization domains, such as those from GCN4 from yeast fibritin from bacteria phage T4 and aspartate transcarbamoylase of Escherichia coli, have been described previously to allow trimerization of heterologous proteins, none of these trimerizing proteins are human in nature, nor are they naturally secreted proteins. As such, any trimeric fusion proteins would have to be made intracellularly, which not only may fold incorrectly for naturally secreted proteins such as soluble receptors, but also make purification of such fusion proteins from thousands of other intracellular proteins difficult. Moreover, the fatal drawback of using such non-human protein trimerization domains (e.g. from yeast, bacteria phage and bacteria) for trimeric biologic drug design is their presumed immunogenicity in the human body, rendering such fusion proteins ineffective shortly after injecting them into the human body.
One secreted protein previously used as a protein trimerization tag is tetranectin, which is a plasminogen-binding protein of C-lectin family. However, unlike IgG Fc dimerization tag, the trimeric tetranectin structure is not strengthened by any interchain disulfide bonds, and significant fractions of both monomeric and dimeric tetranectin co-existed with the trimeric structure in solution. Physiologically, teranectin is involved in tissue remodeling and increased cell matrix concentration of tetranectin in human has been linked to multiple cancer types. Recombinant heterologous tetranectin fusion proteins have only been produced intracellularly in E. coli as insoluble inclusion bodies that required refolding to obtain soluble structures. These unfavorable attributes suggest that tetranectin is not ideal for therapeutic applications as a protein trimerization tag. Nonetheless, bacterially produced ApoAl-Tetranectin fusion protein has been produced and patented and is being tested as a therapeutic agent for atherosclerosis.